Polyvalent immunogen

ABSTRACT

The present invention relates, generally, to a polyvalent immunogen and, more particularly, to a method of inducing neutralizing antibodies against HIV and to a polyvalent immunogen suitable for use in such a method.

[0001] This is a continuation-in-part of application Ser. No.10/373,592, filed Feb. 26, 2003, which is a continuation-in-part ofapplication Ser. No. 10/289,228, filed Nov. 7, 2002, which claimspriority from Provisional Application No. 60/331,036, filed Nov. 7,2001, the contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates, generally, to a polyvalentimmunogen and, more particularly, to a method of inducing neutralizingantibodies against HIV and to a polyvalent immunogen suitable for use insuch a method.

BACKGROUND

[0003] Immunogenic peptides have been developed that elicit B and T cellresponses to various strains of human immunodeficiency virus (HIV)(Palker et al, J. Immunol. 142:3612-3619 (1989), Haynes et al, Trans.Am. Assoc. Physician 106:31-41 (1993), Haynes et al, J. Immunol.151:1646-1653 (1993), Haynes et al, AID Res. Human Retroviruses11:211-221 (1995)) (see also WO 97/14436). These peptides consist of twocomponents, each derived from noncontiguous regions of the HIV gp120envelope protein. One envelope component consists of 16 amino acidresidues from the fourth constant (C4) domain of HIV gp120, and includesa T-helper epitope (Cease et al, Proc. Natl. Acad. Sci. USA 84:4249-4253(1987)). Linked to the carboxyl terminus of this gp120 C4 region peptideis a 23 amino acid segment from the third variable (V3) domain of gp120,that includes a B cell neutralizing antibody epitope for cellline-adapted HIV strains (Palker et al, J. Immunol. 142:3612-3619(1989), (Palker et al, Proc. Natl. Acad. Sci. USA 85:1932-1936 (1988),Rusche et al, Proc. Natl. Acad. Sci. USA 85:3198-3202)), a T-helperepitope (Palker et al, J. Immunol. 142:3612-3619 (1989)), and twocytotoxic T lymphopoietic (CTL) epitopes (Clerici et al, J. Immunol.146:2214-2219 (1991), Safrit et al, 6^(th) NCVDG Meeting, Oct. 30 toNov. 4, 1993)). In mice and rhesus monkeys, these C4-V3 hybrid peptideshave induced antibodies that bind to native gp120 and neutralize theparticular cell line-adapted strain of HIV from which the V3 segment wasderived, as well as induce T helper cell proliferative responses and MHCClass I-restricted CTL responses that kill HIV or HIV protein expressingtarget cells (Palker et al, J. Immunol. 142:3612-3619 (1989), Haynes etal, AID Res. Human Retroviruses 11:211-221 (1995)). Recently, it wasshown that this gp120 peptide design can induce antibodies thatneutralize primary HIV isolates and simian-human immunodeficiencyviruses (SHIV) expressing primary HIV isolate envelopes (Liao et al, J.Virol. 74:254-263 (2000)). Moreover, in a challenge trial of thisimmunogen in rhesus monkeys, it was shown that C4-V3 peptides from thegp120 of the pathogenic SHIV 89.6P, induced neutralizing antibodies thatprevented the fall in CD4 counts after challenge with SHIV 89.6P (Letvinet al, J. Virol. 75:4165-4175 (2001)). Therefore, anti-V3 antibodies canprotect primates against primary isolate SHIV-induced disease.

[0004] A prototype polyvalent HIV experimental immunogen comprised ofthe conserved C4 region of gp120 and the V3 regions of HIV isolates MN,CANO(A), EV91 and RF has been constructed and has been found to behighly immunogenic in human clinical trials (Bartlett et al, AIDS12:1291-1300 (1998), Graham et al, Abstract, AIDS Vaccine (2001)). Thus,understanding secondary and higher order structures of the components ofthis polyvalent immunogen, as well as defining strategies to optimizegp120 immunogen antigenicity, is important to HIV vaccine designefforts. In addition, recent data suggest that the HIV V3 region may beinvolved in regulating gp120 interactions with HIV co-receptors, CXCchemokine receptor 4 (CXCR4) and chemokine receptor type 5 (CCR5)(Berger, AIDS Suppl. A:53-56 (1997)).

[0005] In previous studies, nuclear magnetic resonance (NMR) has beenused to characterize conformations of the multivalent immunogen C4-V3peptides in solution (de Lorimier et al, Biochemistry 33:2055-2062(1994), Vu et al, Biochemistry 35:5158-5165 (1996), Vu et al, J. Virol.73:746-750 (1999)). It as been found that the V3 segments of each of thefour C4-V3 peptides displayed evidence of preferred solutionconformations, with some features shared, and other features differingamong the four peptides. The C4 segment, which is of identical sequencein all the peptides, showed in each case a tendency to adopt nascenthelical conformations (de Lorimier et al, Biochemistry 33:2055-2062(1994), Vu et al, Biochemistry 35:5158-5165 (1996), Vu et al, J. Virol.73:746-750 (1999)).

[0006] The C4 sequence as a peptide does not elicit antibodies that bindnative gp120 (Palker et al, J. Immunol. 142:3612-3619 (1989), Haynes etal, J. Immunol. 151:1646-1653 (1993), Ho et al, J. Virol. 61:2024-2028(1987), Robey et al, J. Biol. Chem. 270:23918-23921 (1995)). This led tothe speculation that the nascent helical conformations exhibited by theC4 segment might reflect a conformation not native to HIV gp120.Amino-acid sequence homology between the gp120 C4 region and a human IgACH1 domain has been noted (Maddon et al, Cell 47:333-348 (1986)). Bycomparison to the structure of mouse IgA (Segal et al, Proc. Natl. Acad.Sci. USA 71:4298-4302 (1974)), the C4-homologous region of IgA has a βstrand secondary structure (de Lorimier et al, Biochemistry 33:2055-2062(1994)). Therefore, while the C4 gp120 peptide in solution adoptsnascent helical conformations, the native structure of this gp120 C4region may be quite different (ie, in the context of gp 120 have a βstrand secondary structure).

[0007] The present invention results, at least in part, from the resultsof a study with a three-fold purpose. First, C4-V3HIVRF peptides withamino acid substitutions designed to minimize C4 α-helical peptideconformation and promote β strand C4 secondary structures wereconstructed in order to induce anti-native gp120 antibodies with themodified C4 peptide. Second, tests were made to determine if any ofthese mutated C4-V3RF peptides would enhance gp120 V3 region peptideimmunogenicity, and therefore augment anti-HIVRF gp120 V3 loop antibodyresponses. Finally, the solution conformers of each peptide studiedimmunologically were also solved using NMR to correlate peptideconformers with peptide immunogenicity.

SUMMARY OF THE INVENTION

[0008] The present invention relates to a method of inducingneutralizing antibodies against HIV and to peptides, and DNA sequencesencoding same, that are suitable for use in such a method.

[0009] Objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1: Summary of antibody binding titers to immunizing peptideafter 2 or 3 boosts of 3 mice in each group with immunizing peptide.There was a slight enhancement of levels of antibody induced by the E9Gvariant after 2 but not 3 boosts, while the E9V variant significantlyboosted antibody levels compared to the C4-V3RF(A) peptide after 2 and 3boosts. Antibody to the K12E variant induced by the K12E peptide wassignificantly lower than C4-V3RF(A) induced antibody levels after both 2and 3 boosts.

[0011]FIG. 2: NMR spectra of the four C4-V3RF variant peptides.

[0012]FIG. 3: C4_(E9V)-V389.6 peptides bound better to human PBlymphocytes and monocytes than did the C4-V389.6 peptides. Similar datawere obtained with the C4-V389.6P and C4-E9V-89.6P peptides. Sequence ofthe C4-V389.6 peptide form HIV89.6 isolate was:KQIINMWQEVGKAMYA-TRPNNNTRRRLSIGPGRAFYARR; the sequence of theC4_(E9V)-V389.6 peptide was: KQIINMWQVVGKAMYA-TRPNNNTRRRLSIGPGRAFYARR;the sequence of the C4-V389.6P peptide was:KQIINMWQEVGKAMYA-TRPNNNTRERLSIGPGRAFYARR; the sequence of theC4E9V-V389.6P peptide was: KQIINMWQVVGKAMYA-TRPNNNTRERLSIGPGRAFYARR.

[0013]FIG. 4: Neutralization of BAL in PBMC.

[0014]FIG. 5: Neutralization of HIV primary isolates by sera from guineapig (GP) 469 immunized with the C4-V3 peptide 62.19. The isolates testedare listed on the right side. The grey and white areas indicate noneutralization. The red boxes indicate >50% neutralization. The titersare 1:10, 1:30, 1:90 and 1:270 going across in each column.

[0015]FIG. 6: C4-V3 sequences tested.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to a composition comprising amultiplicity of immunogenic hybrid peptides, each comprising twocomponents. One component includes a T-helper epitope and can compriseresidues from the C4 domain of HIV gp120. The second component comprisesresidues from the V3 domain of gp120 and includes a B cell neutralizingantibody epitope.

[0017] Advantageously, the first component comprises about 16 contiguousresidues from the C4 domain (about residues 421 to 436) and the secondcomponent comprises about 23-25 contiguous residues from the V3 domain(about residues 297 to 322). The components can, however, be longer, andcan comprise, for example, the entirety of the cysteine to cysteine V3loop region, or be shorter. Preferably, the V3 component is linked Cterminal to the C4 component peptide. The hybrid peptides can includeadditional sequences (e.g., linkers (e.g., cysteine, serine or lysinelinkers) between the C4 and V3 components). The composition can, forexample, comprise 5 to 10 hybrid peptides, 10 to 15 hybrid peptides or25 to 30 hybrid peptides. The number of hybrid peptides used willdepend, at least in part, on the target population.

[0018] Preferred first components comprising residues from the C4 domainare shown in the Tables that follow (see particularly Tables 6 and 7).Other T helper determinants from HIV or from non-HIV proteins can alsobe used.

[0019] For example, a further T helper epitope suitable for use in theinvention is from HIV gag (e.g., residues 262-278). One such sequence,designated GTH1, is YKRWIILGLNKIVRMYS (from HIV p24 gag). Variants ofthis sequence can also be used. Alternatively, or in addition, acarbohydrate such as the outer membrane protein of pneumococcus, oranother carbohydrate or protein with immunogenic, T helper activity canbe used.

[0020] The V3 components of the hybrid peptides present in the instantcomposition are selected so as to be representative of higher orderstructural motifs present in a population, which motifs mediate V3functions in the course of envelope mediated HIV interaction with hostcells. The Los Alamos National Laboratories Human Retroviruses and AIDSDatabase (Human Retroviruses and AIDS, 2000, Published by theTheoretical Biology and Biophysics G T-10, Mail Stop K710, LANL, LosAlamos, N. Mex.) presently contains over 14,000 HIV V3 envelopesequences, showing the extraordinary diversity the virus has obtainedsince originating in man in Africa approximately 50 years ago. Forexample, among 432 HIV-1 V3 sequences derived from individuals infectedwith subtype C (designated “Clade C”) in Africa currently available inthe HIV database, 176 distinct variants of a 23 amino acid stretch atthe tip of the V3 loop have been found. Similarly, among 6870 B subtype(designated “Clade B”) V3 sequences from the US, 1514 unique forms havebeen found.

[0021] A method has been developed to organize short antigenic domainsby protein similarity scores using maximum-linkage clustering. Thismethod enables the visualization of the clustering patterns as adendrogram, and the splitting patterns in the dendrogram can be used todefine clusters of related sequences (Korber et al, J. Virol.68:6730-6744 (1994)). The method allows the use of several differentamino acid similarity scoring schemes available in the literature,preferred is the amino acid substitution matrix developed by Henikoffand Henikoff (see Advances in Protein Chemistry 54:73-97 (2000) andProteins: Structure, Function and Genetics 17:49-61 (1993)), designed togive substitutions that are well tolerated in conserved proteinstructural elements a high score, and a low score to those that are not.Typically excluded from consideration very rare, highly divergentpeptides, and favored are peptides found in many individuals within thepopulation. In a selected set of sequences, most of the unique forms arewithin one or two amino acids is from a least one other of the peptideschosen. This method has been applied to clustering the large number ofvariants of the antigenic tip of the V3 domain within Clade B and CladeC into groups (about 25) that are likely to be cross-reactive within thegroup. Based on these clustering patterns, variants (e.g., about 25-30)are selected that are representative or “central” to each group, fortesting for antigenicity. The HIV Clade B and Clade C gp120 envelope V3sequences have been analyzed, as described above, for groups of V3sequences predicted to have structural similarities. Twenty five Clade Cand 30 Clade B groups have been defined, and chosen out of each group isa common, or the most common, sequence as a representative of thatgroup. The selected V3 sequences have been included in a C4-V3 designthereby providing a 25 peptide Clade C immunogen, and a 30 peptide CladeB immunogen (see Tables 6 and 7). TABLE 6 C4-V3 design of Clade C V3sequences C4-V3-C1 KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfyatg C4-V3-C2KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfyaRg C4-V3-C3KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfyaAg C4-V3-C4KQIINMWQVVGKAMYA-IrpnnntrksVrigpGqtfyatg C4-V3-C5KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfFatg C4-V3-C6KQIINMWQVVGKAMYA-trpnnntrksirigpGqtfyatN C4-V3-C7KQIINMWQVVGKAMYA-trpnnntrEsirigpGqtfyatg C4-V3-C8KQIINMWQVVGKAMYA-trpnnntrRsirigpGqAfyatg C4-V3-C9KQIINMWQVVGKAMYA-trpnnntrkGirigpGqtfyatg C4-V3-10KQIINMWQVVGKAMYA-trpSnntrksirigpGqAfyatg C4-V3-C11KQIINMWQVVGKAMYA-trpSnntrksirigpGqtfyatN C4-V3-C12KQIINMWQVVGKAMYA-trpSnntrEsirigpGqtfyatg C4-V3-C13KQIINMWQVVGKAMYA-trpnnntrksMrigpGqtfyatg C4-V3-C14KQIINMWQVVGKAMYA-trpGnntrksMrigpGqtfyatg C4-V3-C15KQIINMWQVVGKAMYA-trpGnntrksirigpGqtLyatg C4-V3-C16KQIINMWQVVGKAMYA-VrpnnntrksVrigpGqtSyatg C4-V3-C17KQIINMWQVVGKAMYA-trpGnntrRsirigpGqtfyatg C4-V3-C18KQIINMWQVVGKAMYA-IrpGnntrksVrigpGqtfyatg C4-V3-C19KQIINMWQVVGKAMYA-trpnnntrksirigpGqAfyatN C4-V3-C20KQIINMWQVVGKAMYA-trpnnntrQsirigpGqAfyatK C4-V3-C21KQIINMWQVVGKAMYA-trpGnntrksirigpGqAfFatg C4-V3-C22KQIINMWQVVGKAMYA-trpGnntrksVrigpGqAfyatN C4-V3-C23KQIINMWQVVGKAMYA-trpnnntrkGiHigpGqAfyaAg C4-V3-C24KQIINMWQVVGKAMYA-trpnnntrkGiGigpGqtfFatE C4-V3-C25KQIINMWQVVGKAMYA-trpGnntrEsiGigpGqAfyatg

[0022] TABLE 7 C4-V3 peptides Clade B C4-V3-396.2KQIINMWQVVGKAMYAA-RPNNNTRRNIHGLGRRFYAT-* C4-V3-170.6KQIINMWQVVGKAMYAA-RPNNNTRRSVRIGPGGAMFRTG* C4-V3-82.15KQIINMWQVVGKAMYAA-RPNNNTRRSIPIGPGRAFYTTG* C4-V3-144.8KQIINMWQVVGKAMYAA-RPDNNTVRKIPIGPGSSFYTT-* C4-V3-23.38KQIINMWQVVGKAMYAA-RPIKIERKRIPLGLGKAFYTTK* C4-V3-365.2KQIINMWQVVGKAMYAA-RPSNNTRKGIHLGPGRAIYATE* C4-V3-513.2KQIINMWQVVGKAMYAA-RPSNNTRKGIHMGPGKAIYTTD* C4-V3-1448.1KQIINMWQVVGKAMYAA-RPGNTTRRGIPIGPGRAFFTTG* C4-V3-69.18KQIINMWQVVGKAMYAA-RPNNNTRKSIRIGPGRAVYATD* C4-V3-146.8KQIINMWQVVGKAMYAA-RPGNNTRRRISIGPGRAFVATK* C4-V3-113.1KQIINMWQVVGKAMYAA-RPNNNTRRSIHLGMGRALYATG-* C4-V3-51.23KQIINMWQVVGKAMYAA-RPSNNTRRSIHMGLGRAFYTTG-* C4-V3-72.18KQIINMWQVVGKAMYAA-RPNNNTRKGINIGPGRAFYATG-* C4-V3-36.29KQIINMWQVVGKAMYAA-RPNNNTRKGIHIGPGRTFFATG-* C4-V3-70.18KQIINMWQVVGKAMYAA-RPNNNTRKRIRIGHIGPGRAFYATG* C4-V3-89.14KQIINMWQVVGKAMYAA-RPSINKRRHIHIGPGRAFYAT-* C4-V3-163.7KQIINMWQVVGKAMYAA-RLYNYRRKGIHIGPGRAIYATG* C4-V3-57.20KQIINMWQVVGKAMYAA-RPNRHTGKSIRMGLGRAWHTTR* C4-V3-11.85KQIINMWQVVGKAMYAA-RPNNNTRKSINIGPGRAFYTTG---* C4-V3-34.29KQIINMWQVVGKAMYAA-RPNNNTRKSIQIGPGRAFYTTG---* C4-V3-1.481KQIINMWQVVGKAMYAA-RPNNNTRKSIHIGPGRAFYTTG---* C4-V3-85.15KQIINMWQVVGKAMYAA-RPNNNTRKSIHIAPGRAFYTTG---* C4-V3-62.19KQIINMWQVVGKAMYAA-RPNNNTRKSIHIGPGRAFYATE------* C4-V3-125.9KQIINMWQVVGKAMYAA-RPNNNTRRRISMGPGRVLYTTG* C4-V3-35.29KQIINMWQVVGKAMYAA-RPNNNTRKRISLGPGRVYYTTG* C4-V3-74.17KQIINMWQVVGKAMYAA-RPNNNTRKRMTLGPGKVFYTTG* C4-V3-46.26KQIINMWQVVGKAMYAA-RPDNTIKQRIIHIGPGRPFYTT-* C4-V3-122.9KQIINMWQVVGKAMYAA-RPNYNETKRIRIHRGYGRSFVTVR* C4-V3-162.7KQIINMWQVVGKAMYAA-RPGNNTRGSIHLHPGRKFYYSR* C4-V3-3.323KQIINMWQVVGKAMYAA-RPNNNTRKSINMGPGRAFYTTG

[0023] While the above is offered by way of example, it will beappreciated that the same analyses can by performed for HIV Clades A, D,E, F, G, H, M, N, O, etc, to design V3 immunogens that react with HIVprimary isolates from these Clades.

[0024] In addition to the sequences described in Tables 6 and 7, asubstitution has been made in the C4 sequence at position 9 from E to Vto enhance the binding of the C4 region to human immune cell membranes,and to increase immunogenicity (see Example that follows). SubstitutingV for E at position 9 of C4 results in the C4-E9V-V3RF(A) peptideinducing 2-3 logs higher anti-gp120 V3 region antibody levels comparedwith the original C4-V3RFA(A) peptide. The effect of the E9Vsubstitution is not species specific. While not wishing to be bound bytheory, the data may indicate that the ability of the E9V variantpeptide to enhance B cell antibody production is not MHC specific butrather it relates in some manner to non-MHC specific factors, such is asthe ability of the peptides to bind to the lipid bilayer of immunecells. The data presented in FIG. 3 demonstrate the ability ofC4_(E9V)-V389.6 peptides to bind to human PB lymhocytes and monocytes.The ability of the C4 and C4E9V “T helper” determinants to facilitateimmunogenicity of the V3 region may be due to the ability of helicalamphipathic structures to interact with lipid bilayers in a non-MHCrelated manner and promote peptide internalization. The inventionencompasses the use of C4 sequences in addition to those-describedabove.

[0025] In addition to the composition described above, the inventionencompasses each of the hybrid peptides disclosed as well as each of thecomponents (C4 and V3), alone or in covalent or non-covalent associationwith other sequences, as well as nucleic acid sequences encoding any andall such peptides. The invention provides an HIV immunogen that caninduce broadly reactive neutralizing antibodies against HIV of multiplequasispecies, and across clades. With reference to Example 3, the “dualD” HIV isolate, neutralized by serum from GP 469 immunized with peptide62.19 to a titer of 1:30, is a Clade A/G recombinant HIV isolate. Thisdemonstrates that this peptide (62.19), for example, can induceantibodies against a non-B HIV isolate. The 62.19 and other V3 sequencesin FIG. 6 and Tables 10, 11 and 12 can be expressed either alone or, forexample, as a C4-V3 sequence, as in FIG. 6. It will be appreciated thatthe same analysis described in Example 3 can by performed for any of HIVClades A, D, E, F, G, H, M, N, O, etc, to identify V3 immunogens thatreact with HIV primary isolates from one or more of these Clades.

[0026] The peptide immunogens of the invention can be chemicallysynthesized and purified using methods which are well known to theordinarily skilled artisan. (See, for example, the Example thatfollows.) The composition can comprise the peptides linked end to end orcan comprise a mixture of individual peptides. The peptide immunogenscan also be synthesized by well-known recombinant DNA techniques.Recombinant synthesis may be preferred when the peptides are covalentlylinked. Nucleic acids encoding the peptides of the invention can be usedas components of, for example, a DNA vaccine wherein the peptideencoding sequence(s) is/are administered as naked DNA or, for example, aminigene encoding the peptides can be present in a viral vector. Theencoding sequence(s) can be present, for example, in a replicating ornon-replicating adenoviral vector, an adeno-associated virus vector, anattenuated mycobacterium tuberculosis vector, a Bacillus Calmette Guerin(BCG) vector, a vaccinia or Modified Vaccinia Ankara (MVA) vector,another pox virus vector, recombinant polio and other enteric virusvector, Salmonella species bacterial vector, Shigella species bacterialvector, Venezuelean Equine Encephalitis Virus (VEE) vector, a SemlikiForest Virus vector, or a Tobacco Mosaic Virus vector. The encodingsequence(s), can also be expressed as a DNA plasmid with, for example,an active promoter such as a CMV promoter. Other live vectors can alsobe used to express the sequences of the invention. Expression of theimmunogenic peptides of the invention can be induced in a patient's owncells, by introduction into those cells of nucleic acids that encode thepeptides, preferably using codons and promoters that optimize expressionin human cells. Examples of methods of making and using DNA vaccines aredisclosed in U.S. Pat. Nos. 5,580,859, 5,589,466, and 5,703,055.

[0027] The composition of the invention comprises an immunologicallyeffective amount of the peptide immunogens of this invention, or nucleicacid sequence(s) encoding same, in a pharmaceutically acceptabledelivery system. The compositions can be used for prevention and/ortreatment of immunodeficiency virus infection. The compositions of theinvention can be formulated using adjuvants, emulsifiers,pharmaceutically-acceptable carriers or other ingredients routinelyprovided in vaccine compositions. Optimum formulations can be readilydesigned by one of ordinary skill in the art and can includeformulations for immediate release and/or for sustained release, and forinduction of systemic immunity and/or induction of localized mucosalimmunity (e.g, the formulation can be designed for intranasaladministration). The present compositions can be administered by anyconvenient route including subcutaneous, intranasal, oral,intramuscular, or other parenteral or enteral route. The immunogens canbe administered as a single dose or multiple doses. Optimum immunizationschedules can be readily determined by the ordinarily skilled artisanand can vary with the patient, the composition and the effect sought. Byway of example, it is noted that approximately 50 μg-100 μg of eachhybrid peptide can be administered, for example, intramuscularly (e.g.3×).

[0028] The invention contemplates the direct use of both the peptides ofthe invention and/or nucleic acids encoding same and/or the peptidesexpressed as minigenes in the vectors indicated above. For example, aminigene encoding the peptides can be used as a prime and/or boost.Importantly, it has been recently shown that recombinant gp120 is notefficacious as a vaccine for HIV in phase III trials (Elias, P., DurhamMorning Herald, Feb. 25, 2003; VaxGen News Conference, Feb. 24, 2003).Thus, it would be advantageous to express, for example, the 62.19 V3loop and/or other V3 loops in Table 11 or 12 in the context of gp120molecules or gp160 or gp140 molecules, either as expressed solublerecombinant proteins, or expressed in the context of one of the vectorsdescribed above. This strategy takes advantage of the ability to expressnative V3 conformations within a whole gp120 or gp140 or gp160 HIVenvelope protein.

[0029] One of the preferred gp120, gp140 or gp160 envelopes that, forexample, 62.19 V3 loops can be expressed with is that of consensus orancestral HIV envelope artificial sequences (Gaaschen et al, Science296:2354-2360 (2002)). Although artificial and computer designed, onesuch sequence (the consensus of consensus envelope) gp120 (con 6) hasbeen shown to bind soluble CD4 and anti-gp120 mabs A32, 1b12, 2G12.After binding mab A32 or soluble CD4, the con 6 gp120 binds the CCR5binding site mab 176—indicating a “native” gp120 conformation.

[0030] Thus, the entire V3 loops from the Los Alamos Database from thesequences of one or more of the peptides in Table 11 or 12 can beexpressed in the consensus (con 6) or other consensus or ancestralgp120, gp140, or gp160 envelope protein, or expressed in a native gp120,gp140, or gp160, such as HIV BAL or HIV JRFL, and used as an immunogenas a recombinant envelope protein, or used as an immunogen expressed inone of the vectors above.

[0031] The V3 peptides or recombinant proteins can be used as primes orboosts with the V3 peptides or recombinant gp120s, gp140s or gp160sexpressed in the above vectors used as primes or boosts.

[0032] A preferred immunogen is the consensus 6 gp120 expressing thefull-length 62.19 V3 loop, expressed as a DNA plasmid as a primaryimmunization, followed by adenovirus expressing the Con 6 envelopeexpressing the 62.19 V3 sequence from the Los Alamos Database as abooster immunization.

[0033] Certain aspects of the invention can be described in greaterdetail in the non-limiting Example that follows.

EXAMPLE 1

[0034] Experimental Details

[0035] Peptide Design, Synthesis and Purification.

[0036] Peptides were designed, as shown in Table 1. It was hypothesizedthat alteration of the C4 sequence to reduce its helical conformationaltendency in peptides might cause enrichment of solution conformersresembling a β strand conformation. This in turn might cause C4 to beimmunogenic for antibodies recognizing the native conformation of the C4(part of the CD4 binding site) region of gp120. The present workdescribes tests of this hypothesis in chimeric peptide C4-V3 RF, whichhas a V3 segment from gp120 of HIV strain RF, and three sequencevariants wherein single amino-acid replacements have been introduced atposition 9 in the C4 segment, Glu (E) to Gly (G), Glu (E) to Val (V),and at position 12, Lys (K) to Glu (E) (Table 1). These replacementswere made in part to disrupt possible stabilization of helicalconformations due to side-chain (i, i+3) charge interaction between E9and K12 (Scholtz et al, Biochemistry 32:9668-9676 (1993)). In addition,the substitution in C4_(E9G)-V3RF(A) was expected to disfavor helixformation by introducing greater main-chain flexibility (Chakrabartty etal, Adv. Protein Chem. 46:141-176 (1995)). Furthermore the substitutionin C4_(E9G)-V3RF(A) introduced two adjacent valine residues which hasbeen hypothesized to favor extended conformations. Thus, the parentpeptide, C4-V3RF(A) (Haynes et al, AID Res. Human Retroviruses11:211-221 (1995)) contained 16 N-terminal residues from the C4 domainof gp120_(IIIB) and 23 C-terminal residues from the V3 domain of gp120of HIVRF. TABLE 1 Peptides Used in This Study Sequence Peptide C4 V31             16  17                   39 C4-V3RF(A) KQIINMWQEVGKAMYA TRPNNNTRKSITKGPGRVIYATG C4_(E9G)-V3RF(A) KQIINMWQGVGKAMYA TRPNNNTRKSITKGPGRVIYATG C4_(E9V)-V3RF(A) KQ]INMWQVVGKAMYA TRPNNNTRKSITKGPGRVIYATG C4_(K12E)-V3RF(A) KQIIINMWQEVGEAMYATRPNNNTRKSITKGPGRVIYATG

[0037] Peptides were synthesized by fluorenylmethoxycarbonyl chemistryon an ABI 43 1A peptide synthesizer (Applied Biosystems, Inc., FosterCity, Calif.), then purified by reverse-phase high performance liquidchromatography. The purity and identity of the product were confirmed bydetermining molecular mass by electrospray mass spectrometry.

[0038] Immunization Methods.

[0039] Mice were immunized with 50 μg of the indicated peptide inincomplete Freund's adjuvant (1SA51, Seppic Inc., Paris France) at weeks0, 3, and 7 and bled at weeks 2, (bleed 1 after boost 1), week 5 (bleed2 after boost 2) and week 8 (bleed 3 after boost 3). Immune responseswere seen after bleed 2 in most animals and data are reported frombleeds 2 and 3.

[0040] Guinea pigs were immunized intranasally with 200 μg of C4-V3peptide in saline with 1 g of cholera toxin as adjuvant as described.Guinea pigs were immunized on day 0, day 14 and day 21 and serum samplesbefore and 1 week following each immunization obtained by cardiacpuncture.

[0041] ELISA Assay.

[0042] Anti-HIV env peptide ELISA assays were performed as previouslydescribed (Haynes et al, J. Immunol. 151:1646-1653 (1993), Haynes et al,AID Res. Human Retroviruses 11:211-221 (1995)).

[0043] Splenocyte Proliferation Assay.

[0044] Mouse splenocyte proliferation assay using ³H-thymidineincorporation was performed as previously described (Haynes et al, AIDRes. Human Retroviruses 11:211-221 (1995)).

[0045] Neutralizing Antibody Assays.

[0046] Assays for ability of anti-HIV antisera to neutralize HIV wereperformed as described (Palker et al, J. Immunol. 142:3612-3619 (1989),Haynes et al, Trans. Am. Assoc. Physician 106:31-41 (1993), Haynes etal, J. Immunol. 151:1646-1653 (1993), Haynes et al, AID Res. HumanRetroviruses 11:211-221 (1995)).

[0047] NMR spectroscopy.

[0048] Peptides were dissolved to 4 mM in a solution of 90% ¹H₂O, 10%²H₂O, 20 mM NaCl, 5 mM KH₂PO₄, 1 mM sodium azide, 0.5 mM sodium3-(trimethylsilyl) propionate, at a pH of 4.2. The methyl resonance ofthe latter component served as a chemical shift reference.

[0049] Spectra of samples prepared in this way were acquired with aVarian Unity 500 MHz spectrometer at a temperature of 278 K. The locksignal was from deuterium in the sample. The following two-dimensionalspectra were obtained: (a) double-quantum-filtered correlationspectroscopy (DQF-COSY) (Piantini et al, J. Am. Chem. Soc. 104:6800-6801(1982), Rance et al, Biochem. Biopjys. Res. Commun. 117:479-485 (1983));(b) total correlation spectroscopy (TOCSY) (Bax et al, J. Magn. Reson.65:355-360 (1985), Levitt et al, J. Magn. Reson. 47:328-330 (1982)) witha mixing time of 150 ins; and (c) nuclear Overhauser exchangespectroscopy (NOESY) (Jeener et al, J. Phys. Chem. 71:4546-4553 (1979))with a mixing time of 300 ins. Water resonance was suppressed byselective saturation during the relaxation delay, and, for NOESY, duringthe mixing period. The spectral width was 6700 Hz, with the indirectlyacquired dimension collected as 750 (COSY), 512 (TOCSY), or 350 (NOESY)complex increments; and the directly acquired dimension containing 1024complex points. Data were processed with FELIX 2.3 software (Biosym, SanDiego, Calif.). Directly acquired free-induction decays were correctedfor base-line offset. Decays in both dimensions were multiplied by asinebell-squared function (phase shifted by 75°) and zero-filled to 2048points before Fourier-transformation.

[0050] Peptide Membrane Binding Assay.

[0051] Peptides at 100 ng/ml were incubated with 106 peripheral bloodmononuclear cells for 1 hour at 4° C., washed ×3 with phosphate bufferedsaline PHz 7.0, contained 0.1% sodium azide, then incubated guinea piganti-HIV 89.6 V3 antisera (x1hr) (Liao et al, J. Virol. 74:254-263(2000)), wash as above and then incubated with FITC-conjugated goatanti-guinea pig IgG. After a final wash as alone, the cells wereanalyzed for the relative amount of peptide bound to either PBlymphocytes or PB monocytes as reflected in the mean fluorescent channel(MFC) of reactivity of the anti-HIV 89.6 V3 antisera.

[0052] Results

[0053] Anti-gp120 V3 Antibody Responses Following Immunization of MiceWith C4-V3RF, C4_(E9V)-V3RF(A), C4_(E9G)-V3RF(A) and C4_(K12E)-V3RF(A)Peptides.

[0054] First, the ability of C4-V3HIVRF variants to modulate theimmunogenicity of the peptide with regard to antibodies to the V3portion of the C4-V3 immunogen were assayed. The results (FIG. 1, Table2) show differences among the four peptides in their ability to induceanti-HIVRF V3 antibody responses. Sera from C4_(E9V)-V3RF(A)-immunizedmice had a log higher anti-V3 antibody titer than either mice immunizedwith the native C4-V3RF(A) peptide or the C4_(E9V)-V3RF(A) peptidevariant. After one immunization, no anti-V3RF antibody response was seenin mice immunized with either C4-V3RF(A), C4_(E9G)-V3RF(A), orC4_(K12E)-V3RF(A) peptides. However, after only one immunization with 50μg of the C4_(E9V)-V3 peptide, the geometric mean titer to V3RF(A)peptide was 1:5012 (n=3 mice), with titers of 1:3200, 1:3200 and1:12,800 in each of the three mice tested, respectively. Thus, the E9VC4-V3RF(A) variant induced a higher titer and earlier anti-gp 120 V3antibody responses than the other C4-V3RF(A) peptides tested. After 2boosts, C4_(E9V)-V3RF(A)-immunized mice had 2 logs higher anti-V3antibody responses than did C4-V3RF(A) immunized mice (FIG. 1, Table 2).TABLE 2 Comparison of the Ability of C4-V3 Peptides To Induce HIV gp120Anti-C4 and Anti-V3 Antibodies in Balb/c Mice Geometric Mean TiterNumber of Peptide on Plate in ELISA For Anti-Peptide Antibody AssayPeptide Immunogen Animals C4 V3RF(A) C4-V3RF(A) C4E9G-V3RF(A)C4E9V-V3RF(A) C412EV3RF(A) C4-V3RF(A) 6 2 1,584 2,239 1,195 1,584 1,412C4_(E9G)-V3RF(A) 6 2 6,310 7,079 5,623 3,162 3,548 C4_(E9V)-V3RF(A) 5 14151,356 131,825 87,096 87,096 114,815 C4_(K12E)-V3RF(A) 6 1 8 8 1 3 3

[0055] The C4_(K12E)-V3RF(A) peptide variant induced anti-V3 antibodyresponses 3 logs lower than the C4-V3RF(A) peptide after 2 immunizations(FIG. 1, Table 2). Thus, single amino-acid replacements in the C4 Thelper region had extraordinary effects on immunogenicity of the HIVRFgp120 V3 domain.

[0056] Comparison of the Ability of C4-V3RF(A) Peptides to InduceAnti-HIV gp120 Peptide 3H-Thymidine Incorporation in Splenocytes FromNaive and Peptide-Immunized Mice.

[0057] Next, C4-V3 peptides were tested for their ability to stimulateproliferation of splenocytes from peptide-immunized mice. Balb/c micewere sacrificed after the third peptide immunization and theirsplenocytes assayed for the ability to proliferate to PHA and to eachpeptide type (Table 3). It was found that C4-V3RF(A), C4_(E9V)-V3RF(A),and C4_(K12E)-V3RF(A) peptides all induced in vitro proliferativeresponses to the immunizing peptides, whereas the C4_(E9G)-V3RF(A)variant peptide did not induce proliferative responses in E9G-primedmice significantly over responses of naive mice (Table 3). Regarding theability of the E9V peptide variant to induce earlier and greater anti-V3antibody responses compared to the other peptides tested, theC4_(E9V)-V3RF(A) peptide-primed splenocytes for proliferation to theimmunizing peptide only minimally better than did each of the otherthree peptides (Table 3). Thus, altered induction of T helper cellproliferative responses did not explain the differences in peptideimmunogenicity. TABLE 3 Comparison of the Ability of C4-V3 Peptides ToInduce Anti-HIV gp120 Peptide ³H-Thymidine Incorporation in Splenocytesfrom Naïve and Immunized Mice Mean ± SEM Δ CPM per 10⁶ Splenocytes inCulture Peptide Used As Stimulator in ³H-Thymidine Incorporation AssayPeptide Immunogen N C4 V3RF(A) C4-V3RF(A) C4_(E9G)-V3RF(A)C4_(E9V)-V3RF(A) C4_(K12E)-V3RF(A) None (Naïve 6 613 ± 322 408 ± 140 149± 84  114 ± 85  74 ± 47 187 ± 165 Balb/c) C4-V3RF(A) 6 2,289 ± 1,332 955± 353  8,390 ± 1,424^(a) 8,067 ± 1,728 6,242 ± 1,787 6,198 ± 1,343C4_(E9G)-V3RF(A) 6 408 ± 95  708 ± 325 2,103 ± 1,170  3,559 ± 2,310^(b)988 ± 340 1,101 ± 399   C4_(E9V)-V3RF(A) 5 84 ± 52 1,463 ± 473     933 ±4,528 11,743 ± 3,830  24,824 ± 5,581^(c) 10,269 ± 3,592 C4_(K12E)-V3RF(A) 6 3,430 ± 2,796 4,417 ± 2,217 8,670 ± 3,865 13,237 ±8,563  7,513 ± 2,951 12,644 ± 4,138^(d)

[0058] The lower antibody titer induced by the C4_(K12E)-V3 peptideagainst V3RF(A) was not an artifact attributable to lack of ability ofthe V3 peptide not binding to the ELISA plate, as sera fromC4_(E9V)-V3RF(A)-induced antisera had high reactivity to the V3RF(A)peptide on the ELISA plate. Similarly, the C4_(K12E)-V3RF(A) peptidecould bind anti-V3RF antibody, as multiple antisera raised against C4-V3peptides bound the C4_(K12E)-V3 variant (Table 2).

[0059] Antibody levels to the C4 region were also tested. The C4 regioninduced only a minimal antibody response compared to the V3 region, withall the C4-V3 peptides tested (Table 2).

[0060] Anti-gp120 V3 Antibody Responses Following Immunization of GuineaPigs.

[0061] Next, 2 guinea pigs were immunized each with 200 μg ofC4-V3RF(A), C4_(E9G)-V3 RF(A), C4_(E9V)-V3 RF(A) or C4_(K12E)-V3 RF(A)peptide intranasally with 1 g cholera toxin adjuvant in saline.Intranasal immunization of peptides with cholera toxin has beenpreviously shown to result in CTL and titers of anti-peptide antibodysimilar in levels to titers induced by initial antigens administeredsubcutaneously or intramuscularly in oil in water adjuvants such ascomplete and incomplete Freund's adjuvant. In addition, it was desirableto determine the ability of C4-V3 peptides in an aqueous solution (suchas in saline for intranasal immunization) to induce anti-HIV antibodyresponses in order to correlate reactivity of antibodies generatedagainst peptide in an aqueous adjuvant with peptide conformers solved inan aqueous solution. Finally, there was interest in determining if theamino acid substitutions in the C4 region conferred on the C4-V3peptides the same pattern of immunogenicity as seen in oil in wateradjuvant in mice.

[0062] It was found that after 2 immunizations the C4-V3 RF(A) peptideinduced a mean anti-HIV peptide antibody titer of 3981, peptide inducedtiters of 1 log (GMT=31,623) higher. As in mice, substituting the Glu(E) for Lys (K) at position 12 in the C4 peptide abrogated peptideimmunogenicity in guinea pigs (GMT=16) (Table 4). TABLE 4 Titers ofC4-V3 HIV Envelope Antibodies Induced by C4-V3RF(A) Peptides in GuineaPigs Immunizing Peptide Titer Against Immunizing Peptide* C4-V3RF(A)3,981 C4-_(E9G)-V3RF(A) 2,818 C4-_(E9V)-V3RF(A) 31,623C4-_(K12E)-V3RF(A) 16

[0063] Ability of Antibodies Against C4-V3 Peptides to InduceNeutralizing Antibodies.

[0064] In order to induce high levels of neutralizing antibodies withC4-V3 peptides, usually 5 immunizations are given (Palker et al, J.Immunol. 142:3612-3619 (1989), Haynes et al, J. Immunol. 151:1646-1653(1993), Palker et al, Proc. Natl. Acad. Sci. USA 85:1932-1936 (1988),Liao et al, J. Virol. 74:254-263 (2000)). The guinea pig sera from theexperiment presented in Table 4 were tested for ability to neutralizeHIVRF. It was found that one sera from the C4-V3RF(A)-immunized animals(after 3 injections) had a neutralizing antibody titer of 1:40 againstHIVRF, while one animal of the C4_(E9V)-V3RF(A)-injected animals had aneutralizing titer of 1:340 after only 2 injections. Thus, antibodiesinduced by the C4_(E9V)-V3RF(A) peptide can bind to native gp120 andneutralize HIVRF.

[0065] Inability of the C4-E9 V-RF(A) Sera to Bind to gp120 fromHIV_(IIIB).

[0066] The V3 loop sequence of HIV_(IIIB) is different from that ofHIVRF, and thus HIVRF anti-V3 neutralizing antibodies do not neutralizeHIV_(IIIB). To determine if any antibodies were generated by any of theC4-V3RF(A) variant peptides, all the mouse sera in Table 2 were tested,as were the guinea pig sera in Table 4, for the ability to bind tonative recombinant HIV_(IIIB) gp120 in ELISA. Since anti-HIVRF V3antibodies do not bind to the HIV_(IIIB) V3 loop, any binding activityof these anti-C4-V3 sera would be to the C4 region of HIV_(IIIB), whichis conserved between HIV_(IIIB) and HIVRF. No binding of any mouse orguinea pig anti-C4-V3 sera to HIV_(IIIB) gp120 was seen, indicating theinability of these peptides to induce antibodies against the nativegp120 C4 region.

[0067] Conformational Propensities of C4-V3 RF Sequence Variants inAqueous Solution.

[0068] Next, the peptides were examined by NMR to determine whetherconformational changes had been induced by amino-acid sequencealteration. It was hypothesized that specific amino-acid substitutionsin the C4 segment would lead to a decrease in the tendency of thisregion to adopt transient helical conformations. To test thishypothesis, each of the four peptides, C4-V3RF and variants E9G, E9V andK12E, was subjected to ¹H NMR spectroscopy to assign resonances and toanalyze nuclear Overhauser effects between hydrogen nuclei on separateresidues.

[0069] Resonance assignments for nearly all ¹H were determined fromTOCSY, DQF-COSY, and NOESY spectra by standard methods (Wuthrich, NMR ofProteins and Nucleic Acids, John Wiley and Sons, New York (1986)), andare shown in FIG. 2. The value of the chemical shift for a main-chain¹H, for example, the a carbon C^(a)H, is correlated with secondarystructure in the case of proteins or well structured peptides (Wishartet al, J. Mol. Biol. 222:311-333 (1991)). Hence, strong tendencies amongC4-V3RF peptides to adopt secondary structure in solution may bemanifested in chemical shift values. This was examined by calculatingfor each peptide the difference in chemical shift between the C—H ofeach residue and a shift value representing the average for allsecondary structures in proteins (Wishart et al, J. Mol. Biol.222:311-333 (1991)). In no peptide were there stretches of sequence withhigh or low values of the chemical shift difference that would beevidence of stable secondary structure, for example helix or β strand.

[0070] NMR parameters such as chemical shift and coupling constants areoften insensitive indicators of weak preferences for particularconformations since their values are the average of the entirepopulation, thus obscuring the contribution of a slight bias forpopulating certain conformations. The nuclear Overhauser effect (NOE) isoften more sensitive at revealing conformational propensities because itmay give rise to a unique signal, although weak, on a backgroundconsisting only of random noise. Hence, NOESY spectra of C4 is V3RF andits variants were characterized to identify each signal and evaluate itsrelative intensity. Sequential and medium range NOEs involvingmain-chain NH or CaH are listed in FIG. 2. These NOEs and the possibleconformational propensities they represent are discussed as follows forC4_(E9G)-V3RF(A) and C4_(E9V)-V3RF(A). Variant C4_(K12E)-V3RF(A)K12E isdiscussed separately below because it was studied under differentconditions.

[0071] In terms of overall conformation, all four peptides showed NOEpatterns suggesting no tendency to adopt stable structure. For example,sequential daN(i, i+1) and dNN(i, i+1) NOEs were usually both presentfor each sequential pair of residues, with the former typically moreintense, indicating that f and j main-chain dihedral bond angles variedand maintained on average an extended conformation (Dyson et al, Ann.Rev. Biophys. Chem. 20:519-538 (1991)). Also the absence of long rangeNOEs [(i, i+5) or greater] and the few and generally weak medium-rangeNOEs suggested no significant population of higher order structure.

[0072] However, the fact that some medium range NOEs were detected isevidence of propensity to adopt non-random conformations in certainregions (Dyson et al, Ann. Rev. Biophys. Chem. 20:519-538 (1991)).Although only one mixing time was used for NOESY spectra (300 ins),previous studies of a related C4-V3 RF peptide (de Lorimier et al,Biochemistry 33:2055-2062 (1994)) showed that medium range NOEs werestill observable at shorter (75 and 150 ins) mixing times. Hence, theNOEs indicating medium range interactions are not likely due tospin-diffusion.

[0073] Within the C4 segment C4-V3RF and C4_(E9V)-V3RF(A) showednumerous medium range NOEs which are consistent with a tendency of thisregion to populate nascent helical conformations. The presence ofcontiguous or overlapping daN(i,i+2) NOEs from Trp⁷ to Tyr¹⁵ (C4-V3RF)and from Ile⁴ to Lys¹² (E9V) indicates a propensity for nascent helicalturns in these regions (Dyson et al, Ann. Rev. Biophys. Chem. 20:519-538(1991), Dyson et al, J. Mol. Biol. 201:201-217 (1988)). A dNN(i,i+2) NOEin this region in C4-V3 RF (between Lys¹² and Met¹⁴) is also consistentwith main-chain f and j dihedral angles representative of helical turns(Dyson et al, Ann. Rev. Biophys. Chem. 20:519-538 (1991)). C4-V3 RFshows three consecutive daN(i,i+3) NOEs from residues Val¹⁰ to Tyr¹⁵,which is highly indicative of full helical turns. The presence ofequivalent NOEs in E9V could not be ascertained due to overlap withother NOEs. However both C4-V3RF and E9V show two dab(i,i+3) NOEs,between Val¹⁰ and Ala¹³ and between Ala¹³ and Met¹⁴. This type of NOE isalso highly suggestive of full helical turns in these regions of C4.

[0074] Variant C4_(E9G)-V3RF(A) on the other hand showed no evidence, interms of medium range NOEs, for preferential population of certainconformations in C4. This absence of medium range NOEs was not duemerely to ambiguities caused by signal overlap, because there were atleast five positions where an NOE was unambiguously absent inC4_(E9G)-V3RF(A), but present in the parent peptide C4-V3 RF. Thus, theE to G substitution in the C4 peptide appeared to prevent helicalconformer formation in the peptide.

[0075] In the V3 segment of the three peptides, C4-V3 RF,C4_(E9G)-V3RF(A) and C4_(E9V)-V3RF(A), were medium range NOEs suggestingpreferred solution conformations in certain RE regions. All threepeptides showed evidence of a reverse turn in the sequenceArg¹⁸-Pro¹⁹-Asn²⁰-Asn²¹, where these residues comprised positions 1 to4, respectively, of the turn. The NOE pattern consistent with a reverseturn included a weak dNd(i,i+1) between Arg¹⁸ and Pro¹⁹, undetectableddN(i,i+1) between Pro¹⁹ and Asn²¹, weak dad(i,i+1) between Arg¹⁸ andPro¹⁹, strong daN(i,i+1) between Pro¹⁹ and Asn²⁰, and detectabledaN(i,i+2) between Pro¹⁹ and Asn²¹ (Dyson et al, J. Mol. Biol.201:161-200 (1988)). The detection of the weak dNd(i,i+1) NOE (Arg¹⁸ toPro¹⁹) suggested that a Type I turn may be the preferred conformation(Dyson et al, J. Mol. Biol. 201:161-200 (1988)).

[0076] All three peptides also showed evidence of preferred conformersat the sequence Ser²⁶-Ile²⁷-Thr²⁸-Lys²⁹. There were two consecutivedaN(i,i+2) NOEs, between Ser²⁶ and Thr²⁸ and between 11 e²⁷ and Lys²⁹,as well as medium range NOEs not shown in FIG. 2. The latter included adbN(i,i+2) NOE between Ser²⁶ and Thr²⁸, and a dba(i,i+2) NOE betweenthese same residues. The conformational preferences giving rise to theseNOEs did not fit a typical secondary structure, and suggested an unusualturn that placed the side-chain of Ser²⁶ in close proximity to themain-chain groups of Thr²⁸. This type of conformation has been describedas a kink in the context of a helical region (Osterhout et al,Biochemistry 28:7059-7064 (1989)).

[0077] A third conformational feature in the V3 segments of C4-V3RF,C4_(E9V)-V3RF(A) and C4_(E9G)-V3RF(A) occurred in the sequenceGly³⁰-Pro³¹-Gly³²-Arg³³. In E9G the NOEs between these residuesresembled the pattern described above that was consistent with a reverseturn (Dyson et al, J. Mol. Biol. 201:161-200 (1988)). This included aweak dNd(i,i+1) NOE between Gly³⁰ and Pro³¹, a weak ddN(i, i-I-i) NOEbetween Pro³¹ and Gly³², a weak dad(i,i+1) NOE between Gly³⁰ and Pro³¹,a strong daN(i,i+1) NOE between Pro³¹ and Gly³², and a detectabledaN(i,i+2) NOE between Pro³¹ and Arg³³. In the C4-V3RF peptide, thepattern of (i,i+1) NOE intensities was the same but no daN(i,i+2) NOEwas detected between Pro³¹ and Arg³³. Instead a daN(i,i+2) NOE wasdetected between Gly³⁰ and Gly³². And in C4-E9V V3RF, both daN(i,i+2)NOEs, Gly³⁰ to Gly³² and Pro³ to Arg³³, were detected. These data raisedthe possibility that two independent turn-like conformationalpreferences occurred in this region of V3. The fact that a Pro³¹-Arg³³daN(i,i+2) NOE was unambiguously absent in C4-V3RF, and that adaN(i,I+2) NOE between Gly³⁰ and Gly³² was also unambiguously absent inC4_(E9G)-V3RF(A), in spite of sequence identity in all three peptides,may be related to the weak intensity of these NOEs. Being close to thelevel of noise intensity, there is a possibility that one or both NOEsignals on either side of the spectrum will not be detected, thusdisallowing the given NOE to be scored as such.

[0078] Another region in V3 where conformational preferences could beinferred from NOEs occurs in residues Val³⁴-Ile³⁵-Tyr³⁶. In all threepeptides NOEs were observed between the upfield methyl resonance (˜0.67ppm) of Val ³⁴ and the ring hydrogens, both dH and eH, of Tyr³⁶. WeakerNOEs are also seen between the downfield methyl resonance (˜0.89 ppm) ofVal³⁴ and the ring hydrogens of Tyr³⁶. Further evidence of closeproximity between the side-chains of Val³⁴ and Tyr³⁶ was the fact thatthe two methyl resonances of the former had disparate chemical shifts,compared to Val¹⁰, consistent with a ring-current shift induced by thearomatic side-chain of Tyr. One peptide, C4-V3RF(A) had another NOE inthis region, daN(i,i+2) between Ile³⁵ and Ala³⁷, that was unambiguouslyabsent in the C4_(E9G)-V3RF(A) and C4_(E9V)-V3RF(A) peptides. Thisobservation likely represented a poorly populated conformation, perhapsrelated to that which gives rise to the Val³⁴-Tyr³⁶ side-chaininteraction, or from an independent conformational propensity.

[0079] Substitution of Lys¹² with Glu yielded a poorly immunogenicpeptide (C4_(K12E)-V3RF(A)) that, interestingly had solution propertiesdifferent from the other three peptides studied. Under the conditionsused for NMR studies of other C4-V3 peptides, the solution of theC4_(K12E)-V3RF(A) peptides was highly viscous, and viscosity increasedwith pH in the vicinity of pH 4, implicating ionization of the Glu¹²side-chain in this phenomenon. NMR spectra of K12E at 278 K in aqueousbuffer showed a much lower signal-to-noise ratio than the other threepeptides. Increasing the temperature to 318 K or decreasing the pH to3.5 yielded improved but still inadequate signal. Suitably high signalfor resonance assignment and NOE analysis was obtained at 318 K, pH 3.5,20% v/v trifluoroethanol (d₃). Even under this condition the NOEs forthe C⁴ _(K12E)-V3RF(A) were less intense than for other peptides.

[0080] NOE connectivities in the C4 segment of C4_(K12E)-V3RF(A) (FIG.2) show evidence of nascent helical turns in the region between Ile³ andGly¹¹ as inferred from dNN(i, i+2) and daN(i,i+2) NOEs. The stretch fromVal¹⁰ to Thr¹⁷ has two daN(i, i+3) and two dab(i, i+3) NOEs suggestingthe presence of a significant population with full helical turns. Withinthe V3 segment only two medium range NOEs are observed, both daN(i,i+2).Neither corresponds to NOEs observed in the other three peptides, butboth NOEs involve residues of the Ser²⁶-Ile²⁷-Thr²⁸ sequence, for whichthere is evidence of conformational preferences in the other threepeptides. A dbN(i,i+2) NOE between Ser²⁶ and Thr²⁸, observed inC4_(E9V)-V3RF(A)) and C4_(E9G)-V3RF(A), is also observed in the K12Epeptide. Also observed are NOEs between the side-chains of Val³⁴ and Tyr36. Hence the conformations giving rise to these two features are atleast partially preserved under the solution conditions employed forK12E. Differences in the V3 segment between K12E and all of the otherthree peptides include the absence of detectable daN(i,1+2) NOE betweenPro¹⁹ and Asn²¹ and between Ser²⁶ and Thr²⁸. The failure to detect theseNOEs may be due to the overall weaker signals of this sample, or todepopulation of the relevant conformations by the solution conditions.

EXAMPLE 2

[0081] The peptides in Table 7 have been studied in groups of 5 peptidesas indicated in Table 9, and each group of 5 peptides has been injectedinto each of three guinea pigs in Freund's complete then incompleteadjuvant. After 4 immunizations, the animals were bled, and heatinactivated serum was pooled from each animal or tested separately asindicated in Table 8, for the ability to neutralize HIV. Single numbersper group indicate that the results are those of pooled sera from thegroup. Individual results per animal indicate that each serum was testedindividually. Table 8 shows that all the sera neutralized to varyingdegrees the T cell line adapted HIV isolate MN and poorly neutralizedthe TCLA HIV isolate IIIB. Regarding the rest of the isolates in Table8, all of which are HIV primary isolates (89.6, BAL ADA, SF162, 5768,QH0515, PVO, JRFL, BX08, 6101, SS1196), Group C sera from C4-V3 subtypeB peptides neutralized 4/11 (36%) and Group F sera from subtype Bpeptides neutralized 5/11 primary isolates (45%). FIG. 4 shows that forthe HIV CCR5 utilizing primary isolate, BAL, that the individualpeptides in the 5-valent mixture absorbed out the neutralizing activityagainst HIV BAL to varying degrees, whereas the mixture of all thepeptides completely absorbed out the neutralizing activity. TABLE 8Neutralization Of HIV-1 Isolates By Sera From Guinea Pigs Immunized WithC4-V3 Clade B Peptides Animal Immunogen HIVMN# HIVIIIB# SHIV89.6#SHIV89.6# HIVBAL* ADA* 477 A 2,258 0 96 0 478 A 1,357 0 NA 35 0 0 479 A4,632 68 NA 0 480 B 1358 0 NA 0 481 B 7,774 0 NA 27 84 0 482 B 4,241 062 0 483 C 969 0 112 95 484 C 806 0 20 97 84 0 485 C 542 0 226 80 486 D1,488 0 NA 0 487 D 2,184 0 NA 98 80 0 488 D 575 0 NA 0 489 E 3,223 0 NA88 490 E NA 0 NA 255 0 0 491 E 519 0 NA 81 492 F NA 0 NA NA 493 F 910 0NA 0 91 0 494 F 1,159 35 NA NA Animal SF162* 5768* QH0515* PV0* IRFL*BX08* 6101* SS1196* 477 478 90 0 0 0 0 0 0 85 479 480 481 96 0 0 0 0 0 00 482 483 484 99 0 0 0 0 86 0 0 485 486 487 98 0 0 0 0 94 0 0 488 489490 92 0 0 0 0 0 0 0 491 492 493 84 0 0 0 0 91 94 88 494

[0082] TABLE 9 G. Pig Immunization Protocol Part 2 Immunization with agroup of 5 peptides Peptide Name C4-V3 peptide Peptide Sequence Code GPNo. C4-V3-23.33 KQIINMWQVVGKAMYA-RPIKIERKRIPLGLGKAFYTTK A 477, 478, 479C4-V3-11.85 KQIINMWQVVGKAMYA-RPNNNTRKSINIGPGRAFYTTG A C4-C3-34.29KQIINMWQVVGKAMYA-RPNNNTRKSIQIGPGRAFYTTG A C4-V3-1.481KQIINMWQVVGKAMYA-RPNNNTRKSIHIGPGRAFYTTG A C4-V3-3.323KQIINMWQVVGKAMYA-RPNNNTRKSINMGPGRAFYTTG A C4-V3-51.23KQIINMWQVVGKAMYA-RPSNNTRRSIHMGLGRAFYTTG B 480, 481, 482 C4-V3-36.29KQIINMWQVVGKAMYA-RPNNNTRKGIHIGPGRTFFATG B C4-V3-57.20KQIINMWQVVGKAMYA-RPNRHTGKSIRMGLGRAWHTTR B C4-V3-35.29KQIINMWQVVGKAMYA-RPNNNTRKRISLGPGRVYYTTG B C4-V3-46.26KQIINMWQVVGKAMYA-RPDNTIKQRIIHIGPGRPFYTT B C4-V3-69.19KQIINMWQVVGKAMYA-RPNNNTRKSIRIGPGRAVYATD C 483, 484, 485 C4-V3-72.28KQIINMWQVVGKAMYA-RPNNNTRKGINIGPGRAFYATG C C4-V3-70.18KQIINMWQVVGKAMYA-RPNNNTRKRIRIGHIGPGRAFYATG C C4-V3-62.19KQIINMWQVVGKAMYA-RPNNNTRKSIHIGPGRAFYATE C C4-V3-74.17KQIINMWQVVGKAMYA-RPNNNTRKRMTLGPGKVFYTTG C C4-V3-82.15KQIINMWQVVGKAMYA-RPNNNTRRSIPIGPGRAFYTTG D 486, 487, 488 C4-V3-113.1KQIINMWQVVGKAMYA-RPNNNTRRSIHLGMGRALYATG D C4-V3-89.14KQIINMWQVVGKAMYA-RPSINKRRHIHIGPGRAFYAT D C4-V3-85.15KQIINMWQVVGKAMYA-RPNNNTRKSIHIAPGRAFYTTAG D C4-V3-122.9KQIINMWQVVGKAMYA-RPNYNETKRIRIHRGYGRSFVTVR D C4-V3-170.6KQIINMWQVVGKAMYA-RPNNNTRRSVRIGPGGAMFRTG E 489, 490, 491 C4-V3-146.8KQIINMWQVVGKAMYA-RPGNNTRRRIGIGPGRAFVATA E C4-V3-163.7KQIINMWQVVGKAMYA-RLYNYRRKGIHIGPGRAIYATG E C4-V3-125.9KQIINMWQVVGKAMYA-RPNNNTRRRISMGPGRVLYTTG E C4-V3-162.7KQIINMWQVVGKAMYA-RPGNNTRGSIHLHPGRKFYYSR E C4-V3-396.2KQIINMWQVVGKAMYA-RPNNNTRRNIHIGLGRRFYAT F 492, 493, 494 C4-V3-144.8KQIINMWQVVGKAMYA-RPDNNTVRKIPIGPSSFYTT F C4-V3-365.2KQIINMWQVVGKAMYA-RPSNNTRKGIHLGPGRAIYATE F C4-V3-513.2KQIINMWQVVGKAMYA-RPSNNTRKGIRMGPGKAIYTTD F C4-V3-1448.1KQIINMWQVVGKAMYA-RPGNTTRRGIPIGPGRAFFTTG F

[0083] It is important to be able to use T helper determinants with theV3 portion of the peptides shown in Table 7, both to expand the T helperactivity in the immunogen, and in case any of the T helper peptidesshould be found to have any deleterious effects in the course of humantrials. For example, it has recently been found in vitro that in cultureof HIV and T cells, that the C4 portion of the C4-V3 peptide can augmentHIV induced syncytium formation. However, peptides of this generaldesign have been studied in vitro in HIV-infected humans (AIDS12:1291-1300, 1998) and no subjects developed a ≧10 fold change inplasma HIV RNA levels from baseline. Moreover, the primary use of thesepeptides is as an immunogen in HIV-subjects as a preventive vaccine, andnot in doses that one would consider for therapy, which would be inmilligram amounts daily. A T helper determinant from HIV gag, termedGTH1 with the sequence of Y K R W I I L G L N K I V R M Y S has beenconjugated to the V3 of HIV MN and found to induce anti-HIV MN titers of1:3200. Similarly, GTH1 conjugated to a V3 sequence of a HIV primaryisolate DU179 induced antibodies that neutralized HIV MN (1:192) andneutralized the HIV primary isolate JR-FL (90% p24 reduction in PBMCcultures). Thus, the GTH1 T helper sequence can substitute for the C4sequence in the peptides in Table 7.

[0084] Finally, a panel of monovalent serum from individual guinea pigsimmunized with each of the peptides in Table 7 has been screened.Whereas most of the peptides in the list only induced neutralizingantibodies that neutralized 0 to 6 out of 19 primary isolates, 5peptides were found that neutralized from 14 to 19 out of 19 primaryisolates tested. These peptides were C4-V3 36.29, C4-V3 34.29, C4-V362.19, C4-V3 74.17, and C4-V3 162.7. The sequences of these peptides areall listed in Table 7.

[0085] Thus, sufficient breadth has been observed both in mixtures ofC4-V3 peptides and in select individual peptides for the immunogen to bepractical with regard to induction of neutralizing antibodies againstHIV primary isolates. By performing the same immunization studies withthe similarly designed HIV subtype (clade) C peptides in Table 6, that asimilar immunogen(s) can be developed for HIV subtype C viruses.

[0086] While individual peptides can be used to achieve the breadth ofneutralizing activity needed to protect against HIV primary isolates,advantageously, mixtures of multiple peptides are used, such as thecombination of group C, or group F or the combination of C4-V3 36.29,C4-V3 34.29, C4-V3 62.19, C4-V3 74.17, and C4-V3 162.7 peptidesdescribed above.

EXAMPLE 3 HIV-1 Clade B V3-Based Polyvalent Immunogen

[0087] Anti-HIV gp120 V3 antibodies can neutralize some HIV primaryisolates ((Hioe et al, Internat. Immunology 9:1281 (1997), Liao et al,J. Virol. 74:254 (2000), Karachmarov et al, AIDS Res. Human Retrovirol.17:1737 (2001), Letvin et al, J. Virol. 75:4165 (2001)). The hypothesisfor these studies was that sequence variation found among HIV primaryisolates need not reflect the diversity of HIV serotypes, and antibodiescan cross-react with groups of similar viruses. Data from comparison ofNMR structures of several V3 loops and their immunogenicity patternsindicate that there are conserved higher order structures of the V3 thatare similar in antigenicity regardless of primary amino acidheterogeneity (Vu et al, J. Virol. 73:746 (1999)).

[0088] 1514 unique lade B V3 sequences in the Los Alamos NationalLaboratory HIV Database were analyzed by the following methods. Shortantigenic domains were organized by protein similarity scores usingmaximum-linkage clustering (Korber et al, J. Virol. 68:6730 (1994)).This enabled visualization of clustering patterns as a dendritogram, andthe splitting pattern in the dendritogram could be used to defineclusters of related sequences. This method allows the use of severaldifferent amino acid scoring schemes. The amino acid substitution matrixof Henikoff and Henikoff was used which was designed to give amino acidsubstitutions well tolerated in conserved protein structural elements ahigh score, and those that were not, a low score (Henikoff and Henikoff,Protein Structure Function and Genetics 17:49 (1993)). Based on theseclustering patterns, a variant was selected that was most representativeof each group. Excluded were very rare, highly divergent sequences, andfavored were sequences found in many different individuals. This methodallowed for most of the unique V3 sequences to be within one or twoamino acids from at least one of the peptides in the cocktail. Thus,1514 clade B V3 sequences were clustered into 30 groups. The consensuspeptide of each group was synthesized, purified to homogeneity by HPLCand confirmed to be correct by mass spectrometry. Each peptide wasimmunized into a guinea pig (GP) in Incomplete Freunds Adjuvant (IFA),and each sera was tested after the fifth immunization by a singleinfection cycle neutralization assay preformed by ViroLogics, South SanFrancisco, Calif., or by a fusion from without HIV fusion inhibitionassay using aldrithiol-2 inactivated HIV_(ADA), HIV_(MN) and HIV_(AD8)virons (Rosio et al, J. Virol. 72:7992 (1998)).

[0089] The criteria established for acceptable neutralization of primaryisolates was the ability of a serum to neutralize at least 25% of theHIV primary isolates tested. Using these criteria, 7 peptides were foundthat induced neutralizing antibodies against >25% of isolates tested.One of these peptides, peptide 62.19, neutralized 19/19 HIV primaryisolates tested, even when the criteria were increased to greater than80% neutralization vs. 50% neutralization (see FIG. 5 and Table 11).

[0090] When the sequences of 6 peptides that induced no (0/19)neutralization of the 19 primary HIV isolates were evaluated, it wasfound that they were all unusual sequences at the tip of the V3 loop,with sequences such as GLGR, GPGG, GLGK, GLGL, and GLGR present (seeTable 10). Only 1 of the 19 isolates tested had one of the these V3sequences, a GPGG sequence, that was not neutralized by the serum fromthe GPGG-immunized guinea pig.

[0091] Therefore, one serologic defined group of Clade B HIV isolatesmay be defined by the primary amino acid sequences at the tip of theloop of GLGR, GPGG, GLGK, GLGL. TABLE 10 Sequences of Peptides ThatInduced No Neutralization at 50% Inhibition (All Dilutions) Criteria GPNo. Peptide No. V3 Sequence(s) 447 C4-V3 396.2 RPNNNTRRNIHIGLGRRFYAT 448C4-V3 170.6 RPNNNTRRSVRIGPGGAMFRTG 451 C4-V3 23.38RPIKIERKRIPLGLGKAFYTTK 458 C4-V3 51.23 RPSVNNTRRSIHMGLGRAFYTTG 404 C4-V357.20 RPNRHTGKSIRMGLGLRAWHTTR 432 396.2/170.6RRNIHIGLGRRF    RRSVRIGPGGAM

[0092] TABLE 11 Sequences of Peptides That Best Neutralized Clade BIsolates at 50% Inhibition (All Dilutions) Criteria GP No. Peptide No.V3 Sequence(s) 436 69.18/146.8 RKSIRIGPGRAV   RRRISIGPGRAF 4421.481/85.15 RKSIHIGPGRAF   RKSIHIAPGRAF 460(B) C4-V3 36.29RPNNNTRKGIHIGPGRTFFATG 465(A) C4-V3 11.85 RPNNNTRKSINIGPGRAFYTTG 466(A)C4-V3 34.29 RPNNNTRKSIQIGPGRAFYTTG 467(A) C4-V3 1.481RPNNNTRKSIHIGPGRAFYTTG 469(C) C4-V3 62.19 RPNNNTRKSIHIGPGRAFYATE 472(C)C4-V3 74.17 RPNNNTRKRMTLGPGKVFYTTG 475(E) C4-V3 162.7RPGNNTRGSIHLHPGRKFYYSR

[0093] When the peptide sequences that induced neutralization of >25% ofprimary isolates were examined, it was found that the sequences were allsimilar and were all clustered around the Clade B V3 consensus sequenceof IHIGPGRAFYTTG (see Table 11). However, not all peptides with thistype of sequence induced good neutralizing antibodies—15 peptides hadthis type of sequence and did not induce good neutralizing antibodies.Thus, a “computer guided proteomic screen of the V3 loop” has beenperformed and V3 peptides have been identified that express higher orderconformers that mirror the native functionally active motif of the V3that is both available and capable of being bound by neutralizingantibodies. In particular, peptide 62.19 induced neutralizing antibodiesagainst 19 of 19 HIV isolates. Expression of the consensus B V3sequences in Table 11, and expression of certain of the unusual V3sequences in Table 10, can define a “bivalent” clade B immunogen for useworld wide where those sequences are present in the resident HIVquasispecies, likewise, the sequences shown in Table 12. Table 12 showsfull V3 consensus sequences for the V3 loops of the indicated peptides.By placing these full length V3 loop sequences into a full length HIVenvelope gp120 or gp160/gp140 molecule, the ability of these peptides toinduce neutralizing activity is transferred to the HIV envelopecontaining these sequences. Thus, for example, for the artificallydesigned consensus of consensus HIV envelope with less divergence fromother HIV isolates compared to native HIV envelopes (Gaschen et al,Science 296: 2354-2360 (2002)), inclusion of one of the V3 sequences inTable 12 that has been shown to induce neutralizing activity against HIVprimary isolates would augment the ability of the consensus of consensusartifical envelope to induce neutralizing antibodies. Further,expressing the V3 sequences in Table 12 would augment theirimmunogenicity by combining the V3 with other neutralizing sites on animmunogen (the intact envelope monomer or trimer).

[0094] Immunization with a replicating vector, expressing partial orentire (C to C) segments of these V3 loops, can be used to induce longlasting immunity to HIV. TABLE 12 V3 Consensus Sequence Name of TotalSeq peptide in Database Amino Acid Sequence   1.481  945SVEINCTRPNNNTPKSIHIGPGRAFYTTGEIIGDIRQAHCNISRA  62.19C  952SVEINCTRPNNNTRKSIHIGPGRAFYATERIIGDIRQAHCNISRT  62.19ΔT —SVEINCTRPNNNTRKSIHIGPGRAFYATETTRIIGDIRQAHCNISRT 162.7     11SVEINCTRPGNNTRGSIHLHPGRKFYYSRGIIGDIREHCAINIP 170.6      7SVEINCTRPNNNTRRSVRIGPGGAMPRTGDIIGDIRQAHCNLSRT  34.29    39SIEINCTRPNNNTRKSIQIGPGRAFYTTGEIIGDIRQAHCNLSRA  74.17    94SVEINCTRPNNNTRKRMTLGPGKVFYTTGEIIGDIRKAHCNISRA 396.2      2SVAINCTRRNNNTRRNIHIGLGRRFYATEIIGDTKKADCNISRA  23.38    25SVEINCTRPIKIERKRIPLGLGKAFYTTKQVGDIKQAHC  82.15    86PV8NCTRPNNNTRRSIHIAPGRAFYTTGQIIGDIRRAHCNISRT  57.2     21TVVINCTRPNRHTGKSIRMGLGRAWHTTREIIGDIRKAYCTLNGT  36.29    46SVNINCTRPNNNTRKGIHIGPGRTFFATGDIIGDIRQAHCNLSRT BAL V3     CTRPNNNTRKSIHIGPGRAFYTVGEIIGDIRIQAHC

[0095] All documents cited above are hereby incorporated in theirentirety by reference.

1 84 1 39 PRT Artificial Sequence Description of Artificial SequenceHuman Immunodeficiency Virus 1 Lys Gln Ile Ile Asn Met Trp Gln Glu ValGly Lys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg ArgArg Leu Ser Ile Gly Pro Gly 20 25 30 Arg Ala Phe Tyr Ala Arg Arg 35 2 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 2 Lys Gln Ile Ile Asn Met Trp Gln Val Val Gly LysAla Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Arg Arg LeuSer Ile Gly Pro Gly 20 25 30 Arg Ala Phe Tyr Ala Arg Arg 35 3 39 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 3 Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly LysAla Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Glu Arg LeuSer Ile Gly Pro Gly 20 25 30 Arg Ala Phe Tyr Ala Arg Arg 35 4 39 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 4 Lys Gln Ile Ile Asn Met Trp Gln Val Val Gly LysAla Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Glu Arg LeuSer Ile Gly Pro Gly 20 25 30 Arg Ala Phe Tyr Ala Arg Arg 35 5 17 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 5 Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys IleVal Arg Met Tyr 1 5 10 15 Ser 6 39 PRT Artificial Sequence Descriptionof Artificial Sequence Human Immunodeficiency Virus 6 Lys Gln Ile IleAsn Met Trp Gln Val Val Gly Lys Ala Met Tyr Ala 1 5 10 15 Thr Arg ProAsn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro Gly 20 25 30 Gln Thr PheTyr Ala Thr Gly 35 7 39 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 7 Lys Gln Ile Ile AsnMet Trp Gln Val Val Gly Lys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro AsnAsn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe TyrAla Arg Gly 35 8 39 PRT Artificial Sequence Description of ArtificialSequence Human Immunodeficiency Virus 8 Lys Gln Ile Ile Asn Met Trp GlnVal Val Gly Lys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn ThrArg Lys Ser Ile Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Ala Gly35 9 39 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 9 Lys Gln Ile Ile Asn Met Trp Gln Val Val Gly LysAla Met Tyr Ala 1 5 10 15 Ile Arg Pro Asn Asn Asn Thr Arg Lys Ser ValArg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Gly 35 10 39 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 10 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Phe Ala Thr Gly 35 11 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 11 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Asn 35 12 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 12 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Glu SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Gly 35 13 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 13 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Arg SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Ala Phe Tyr Ala Thr Gly 35 14 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 14 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys GlyIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Gly 35 15 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 15 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Ser Asn Asn Thr Arg Lys SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Asn 35 16 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 16 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Ser Asn Asn Thr Arg Lys SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Asn 35 17 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 17 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Ser Asn Asn Thr Arg Glu SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Gly 35 18 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 18 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys SerMet Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Gly 35 19 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 19 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Gly Asn Asn Thr Arg Lys SerMet Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Gly 35 20 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 20 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Gly Asn Asn Thr Arg Lys SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Leu Tyr Ala Thr Gly 35 21 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 21 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Val Arg Pro Asn Asn Asn Thr Arg Lys SerVal Arg Ile Gly Pro Gly 20 25 30 Gln Thr Ser Tyr Ala Thr Gly 35 22 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 22 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Gly Asn Asn Thr Arg Arg SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Gly 35 23 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 23 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Ile Arg Pro Gly Asn Asn Thr Arg Lys SerVal Arg Ile Gly Pro Gly 20 25 30 Gln Thr Phe Tyr Ala Thr Gly 35 24 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 24 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Ala Phe Tyr Ala Thr Asn 35 25 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 25 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Gln SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Ala Phe Tyr Ala Thr Lys 35 26 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 26 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Gly Asn Asn Thr Arg Lys SerIle Arg Ile Gly Pro Gly 20 25 30 Gln Ala Phe Phe Ala Thr Gly 35 27 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 27 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Gly Asn Asn Thr Arg Lys SerVal Arg Ile Gly Pro Gly 20 25 30 Gln Ala Phe Tyr Ala Thr Asn 35 28 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 28 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys GlyIle His Ile Gly Pro Gly 20 25 30 Gln Ala Phe Tyr Ala Ala Gly 35 29 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 29 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys GlyIle Gly Ile Gly Pro Gly 20 25 30 Gln Thr Phe Phe Ala Thr Glu 35 30 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 30 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Gly Asn Asn Thr Arg Glu SerIle Gly Ile Gly Pro Gly 20 25 30 Gln Ala Phe Tyr Ala Thr Gly 35 31 37PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 31 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Arg Asn IleHis Ile Gly Leu Gly Arg 20 25 30 Arg Phe Tyr Ala Thr 35 32 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 32 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Arg Ser ValArg Ile Gly Pro Gly Gly 20 25 30 Ala Met Phe Arg Thr Gly 35 33 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 33 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Arg Ser IlePro Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Gly 35 34 37 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 34 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asp Asn Asn Thr Val Arg Lys IlePro Ile Gly Pro Gly Ser 20 25 30 Ser Phe Tyr Thr Thr 35 35 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 35 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Ile Lys Ile Glu Arg Lys Arg IlePro Leu Gly Leu Gly Lys 20 25 30 Ala Phe Tyr Thr Thr Lys 35 36 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 36 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Ser Asn Asn Thr Arg Lys Gly IleHis Leu Gly Pro Gly Arg 20 25 30 Ala Ile Tyr Ala Thr Glu 35 37 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 37 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Ser Asn Asn Thr Arg Lys Gly IleHis Met Gly Pro Gly Lys 20 25 30 Ala Ile Tyr Thr Thr Asp 35 38 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 38 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Gly Asn Thr Thr Arg Arg Gly IlePro Ile Gly Pro Gly Arg 20 25 30 Ala Phe Phe Thr Thr Gly 35 39 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 39 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleArg Ile Gly Pro Gly Arg 20 25 30 Ala Val Tyr Ala Thr Asp 35 40 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 40 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Gly Asn Asn Thr Arg Arg Arg IleSer Ile Gly Pro Gly Arg 20 25 30 Ala Phe Val Ala Thr Lys 35 41 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 41 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Arg Ser IleHis Leu Gly Met Gly Arg 20 25 30 Ala Leu Tyr Ala Thr Gly 35 42 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 42 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Ser Asn Asn Thr Arg Arg Ser IleHis Met Gly Leu Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Gly 35 43 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 43 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Gly IleAsn Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Ala Thr Gly 35 44 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 44 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Gly IleHis Ile Gly Pro Gly Arg 20 25 30 Thr Phe Phe Ala Thr Gly 35 45 41 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 45 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Arg IleArg Ile Gly His Ile Gly 20 25 30 Pro Gly Arg Ala Phe Tyr Ala Thr Gly 3540 46 37 PRT Artificial Sequence Description of Artificial SequenceHuman Immunodeficiency Virus 46 Lys Gln Ile Ile Asn Met Trp Gln Val ValGly Lys Ala Met Tyr Ala 1 5 10 15 Arg Pro Ser Ile Asn Lys Arg Arg HisIle His Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Ala Thr 35 47 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 47 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Leu Tyr Asn Tyr Arg Arg Lys Gly IleHis Ile Gly Pro Gly Arg 20 25 30 Ala Ile Tyr Ala Thr Gly 35 48 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 48 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Arg His Thr Gly Lys Ser IleArg Met Gly Leu Gly Arg 20 25 30 Ala Trp His Thr Thr Arg 35 49 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 49 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleAsn Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Gly 35 50 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 50 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleGln Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Gly 35 51 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 51 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleHis Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Gly 35 52 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 52 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleHis Ile Ala Pro Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Gly 35 53 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 53 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleHis Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Ala Thr Glu 35 54 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 54 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Arg Arg IleSer Met Gly Pro Gly Arg 20 25 30 Val Leu Tyr Thr Thr Gly 35 55 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 55 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Arg IleSer Leu Gly Pro Gly Arg 20 25 30 Val Tyr Tyr Thr Thr Gly 35 56 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 56 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Arg MetThr Leu Gly Pro Gly Lys 20 25 30 Val Phe Tyr Thr Thr Gly 35 57 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 57 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asp Asn Thr Ile Lys Gln Arg IleIle His Ile Gly Pro Gly 20 25 30 Arg Pro Phe Tyr Thr Thr 35 58 40 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 58 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Tyr Asn Glu Thr Lys Arg IleArg Ile His Arg Gly Tyr 20 25 30 Gly Arg Ser Phe Val Thr Val Arg 35 4059 38 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 59 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Gly Asn Asn Thr Arg Gly Ser IleHis Leu His Pro Gly Arg 20 25 30 Lys Phe Tyr Tyr Ser Arg 35 60 38 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 60 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Arg Pro Asn Asn Asn Thr Arg Lys Ser IleAsn Met Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Gly 35 61 39 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 61 Lys Gln Ile Ile Asn Met Trp Gln Glu Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys SerIle Thr Lys Gly Pro Gly 20 25 30 Arg Val Ile Tyr Ala Thr Gly 35 62 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 62 Lys Gln Ile Ile Asn Met Trp Gln Gly Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys SerIle Thr Lys Gly Pro Gly 20 25 30 Arg Val Ile Tyr Ala Thr Gly 35 63 39PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 63 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 Thr Arg Pro Asn Asn Asn Thr Arg Lys SerIle Thr Lys Gly Pro Gly 20 25 30 Arg Val Ile Tyr Ala Thr Gly 35 64 40PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 64 Lys Gln Ile Ile Ile Asn Met Trp Gln Glu ValGly Glu Ala Met Tyr 1 5 10 15 Ala Thr Arg Pro Asn Asn Asn Thr Arg LysSer Ile Thr Lys Gly Pro 20 25 30 Gly Arg Val Ile Tyr Ala Thr Gly 35 4065 16 PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 65 Lys Gln Ile Ile Asn Met Trp Gln Val Val GlyLys Ala Met Tyr Ala 1 5 10 15 66 45 PRT Artificial Sequence Descriptionof Artificial Sequence Human Immunodeficiency Virus 66 Ser Val Glu IleAsn Cys Thr Arg Pro Gly Asn Asn Thr Arg Gly Ser 1 5 10 15 Ile His LeuHis Pro Gly Arg Lys Phe Tyr Tyr Ser Arg Gly Ile Ile 20 25 30 Gly Asp IleArg Glu Ala His Cys Ala Ile Asn Ile Pro 35 40 45 67 44 PRT ArtificialSequence Description of Artificial Sequence Human Immunodeficiency Virus67 Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Arg Ser 1 510 15 Val Arg Ile Gly Pro Gly Gly Ala Met Phe Arg Thr Gly Ile Ile Gly 2025 30 Asp Ile Arg Gln Ala His Cys Asn Leu Ser Arg Thr 35 40 68 45 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 68 Ser Ile Glu Ile Asn Cys Thr Arg Pro Asn AsnAsn Thr Arg Lys Ser 1 5 10 15 Ile Gln Ile Gly Pro Gly Arg Ala Phe TyrThr Thr Gly Glu Ile Ile 20 25 30 Gly Asp Ile Arg Gln Ala His Cys Asn LeuSer Arg Ala 35 40 45 69 44 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 69 Ser Val Glu Ile AsnCys Thr Arg Pro Asn Asn Asn Thr Arg Lys Arg 1 5 10 15 Met Thr Gly ProGly Lys Val Phe Tyr Thr Thr Gly Glu Ile Ile Gly 20 25 30 Asp Ile Arg LysAla His Cys Asn Ile Ser Arg Ala 35 40 70 44 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 70 SerVal Ala Ile Asn Cys Thr Arg Arg Asn Asn Asn Thr Arg Arg Asn 1 5 10 15Ile His Ile Gly Leu Gly Arg Arg Phe Tyr Ala Thr Glu Ile Ile Gly 20 25 30Asp Thr Lys Lys Ala Asp Cys Asn Ile Ser Arg Ala 35 40 71 39 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 71 Ser Val Glu Ile Asn Cys Thr Arg Pro Ile LysIle Glu Arg Lys Arg 1 5 10 15 Ile Pro Leu Gly Leu Gly Lys Ala Phe TyrThr Thr Lys Gln Val Gly 20 25 30 Asp Ile Lys Gln Ala His Cys 35 72 45PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 72 Pro Val Glu Ile Asn Cys Thr Arg Pro Asn AsnAsn Thr Arg Arg Ser 1 5 10 15 Ile His Ile Ala Pro Gly Arg Ala Phe TyrThr Thr Gly Gln Ile Ile 20 25 30 Gly Asp Ile Arg Arg Ala His Cys Asn IleSer Arg Thr 35 40 45 73 46 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 73 Thr Val Val Ile AsnCys Thr Arg Pro Asn Arg His Thr Gly Lys Ser 1 5 10 15 Ile Arg Met GlyLeu Gly Arg Ala Val Val His Thr Thr Arg Glu Ile 20 25 30 Ile Gly Asp IleArg Lys Ala Tyr Cys Thr Leu Asn Gly Thr 35 40 45 74 45 PRT ArtificialSequence Description of Artificial Sequence Human Immunodeficiency Virus74 Ser Val Asn Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly 1 510 15 Ile His Ile Gly Pro Gly Arg Thr Phe Phe Ala Thr Gly Asp Ile Ile 2025 30 Gly Asp Ile Arg Gln Ala His Cys Asn Leu Ser Arg Thr 35 40 45 75 36PRT Artificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 75 Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys SerIle His Ile Gly Pro 1 5 10 15 Gly Arg Ala Phe Tyr Thr Thr Gly Glu IleIle Gly Asp Ile Arg Ile 20 25 30 Gln Ala His Cys 35 76 12 PRT ArtificialSequence Description of Artificial Sequence Human Immunodeficiency Virus76 Arg Arg Asn Ile His Ile Gly Leu Gly Arg Arg Phe 1 5 10 77 12 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 77 Arg Arg Ser Val Arg Ile Gly Pro Gly Gly AlaMet 1 5 10 78 12 PRT Artificial Sequence Description of ArtificialSequence Human Immunodeficiency Virus 78 Arg Lys Ser Ile Arg Ile Gly ProGly Arg Ala Val 1 5 10 79 12 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 79 Arg Arg Arg Ile SerIle Gly Pro Gly Arg Ala Phe 1 5 10 80 12 PRT Artificial SequenceDescription of Artificial Sequence Human Immunodeficiency Virus 80 ArgLys Ser Ile His Ile Gly Pro Gly Arg Ala Phe 1 5 10 81 12 PRT ArtificialSequence Description of Artificial Sequence Human Immunodeficiency Virus81 Arg Lys Ser Ile His Ile Ala Pro Gly Arg Ala Phe 1 5 10 82 45 PRTArtificial Sequence Description of Artificial Sequence HumanImmunodeficiency Virus 82 Ser Val Glu Ile Asn Cys Thr Arg Pro Asn AsnAsn Thr Arg Lys Ser 1 5 10 15 Ile His Ile Gly Pro Gly Arg Ala Phe TyrThr Thr Gly Glu Ile Ile 20 25 30 Gly Asp Ile Arg Gln Ala His Cys Asn IleSer Arg Ala 35 40 45 83 45 PRT Artificial Sequence Description ofArtificial Sequence Human Immunodeficiency Virus 83 Ser Val Glu Ile AsnCys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser 1 5 10 15 Ile His Ile GlyPro Gly Arg Ala Phe Tyr Ala Thr Glu Arg Ile Ile 20 25 30 Gly Asp Ile ArgGln Ala His Cys Asn Ile Ser Arg Thr 35 40 45 84 47 PRT ArtificialSequence Description of Artificial Sequence Human Immunodeficiency Virus84 Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser 1 510 15 Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr Glu Thr Thr Arg 2025 30 Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg Thr 35 4045

What is claimed is:
 1. A composition comprising a multiplicity ofimmunogenic peptides comprising a first and a second component, saidfirst component comprising a T-helper epitope, said second componentcomprising residues of the V3 domain of gp120 and including a B cellneutralizing antibody epitope.
 2. The composition according to claim 1wherein said first component is an a human immunodeficiency virus (HIV)T helper epitope.
 3. The composition according to claim 2 wherein saidfirst component comprises residues of the C4 domain of HIV gp120.
 4. Thecomposition according to claim 3 wherein said first component comprisesat least 16 contiguous residues of the C4 domain of HIV gp120.
 5. Thecomposition according to claim 4 wherein said first component comprisesresidues 421 to 436 of the C4 domain of HIV gp120.
 6. The compositionaccording to claim 2 wherein said first component comprises residues ofHIV p24 gag.
 7. The composition according to claim 6 wherein said firstcomponent comprises GTH1 (residues 262-278 of HIV gag).
 8. Thecomposition according to claim 1 wherein said first component is anon-HIV T helper epitope.
 9. The composition according to claim 1wherein said second component comprises at least 23 contiguous residuesof the V3 domain of HIV gp120.
 10. The composition according to claim 9wherein said second component comprises residues 297 to 322 of the V3domain of HIV gp120.
 11. The composition according to claim 1 whereinsaid first component comprises at least 16 contiguous residues of the C4domain of HIV gp120 and said second component comprises at least 23contiguous residues of the V3 domain of HIV gp120.
 12. The compositionaccording to claim 11 wherein said first component comprises residues421 to 436 of the C4 domain of HIV gp120 and said second componentcomprises residues 297 to 322 of the V3 domain of HIV gp120.
 13. Thecomposition according to claim 1 wherein said second component is linkedC terminal to said first component.
 14. The composition according toclaim 1 wherein said first component is linked to said second componentvia a linker.
 15. The composition according to claim 1 wherein saidcomposition comprises at least 5 immunogenic peptides.
 16. Thecomposition according to claim 15 wherein said composition comprisesC4-V3 36.29, C4-V3 34.29, C4-V3 62.19, C4-V3 74.17 and C4-V3 162.7 fromTable
 7. 17. The composition according to claim 15 wherein saidcomposition comprises at least 10 immunogenic peptides.
 18. Thecomposition according to claim 17 wherein said composition comprises atleast 25 immunogenic peptides.
 19. The composition according to claim 1wherein said composition further comprises a carrier.
 20. Thecomposition according to claim 1 wherein said composition furthercomprises an adjuvant.
 21. A composition comprising at least one peptidefrom Table 6 or Table 7 and a carrier.
 22. The composition according toclaim 21 wherein said composition further comprises an adjuvant.
 23. Thecomposition according to claim 1 wherein second components are selectedso as to be representative of higher order structural motifs present ina population, which motifs mediate V3 functions in the course ofenvelope mediated HIV interaction with host cells.
 24. The compositionaccording to claim 23 wherein said composition comprises about 25-30immunogenic peptides the second components of which are selected so asto be representative of infected individuals within a subtype.
 25. Thecomposition according to claim 1 wherein at least one of said firstcomponents comprises the sequence KQIINMWQVVGKAMYA.
 26. A method ofinducing the production of neutralizing antibodies in a patientcomprising administering to said patient an amount of the compositionaccording to claim 1 sufficient to effect said production.
 27. A nucleicacid encoding a peptide in Table 6 or Table
 7. 28. A formulationcomprising at least one nucleic acid sequence encoding said compositionof claim
 1. 29. A method of inducing the production of neutralizingantibodies in a patient comprising administering to said patient anamount of said formulation according to claim 27 sufficient to effectsaid production.
 30. An isolated polypeptide comprising a V3 sequenceshown in Table 10, 11 or
 12. 31. An isolated nucleic acid sequenceencoding at least one polypeptide according to claim
 30. 32. A vectorcomprising the nucleic acid according to claim
 31. 33. A method ofinducing the production of neutralizing antibodies in a mammalcomprising administering to said mammal an amount of said polypeptideaccording to claim 30 or said nucleic acid sequence according to claim31 sufficient to effect said induction.
 34. A composition comprisingsaid polypeptide according to claim 30 or said nucleic acid sequenceaccording to claim 31 and a carrier.
 35. The composition according toclaim 1 wherein said first component comprises the sequenceYKRWIILGLNKIVRM.
 36. The composition according to claim 34 wherein saidpolypeptide is 62.19.
 37. The method according to claim 33 wherein saidmethod comprises administering said V3 sequence in a DNA prime with saidV3 sequence in a gp120 boost.
 37. The method according to claim 33wherein said method comprises administering said V3 sequence in a DNAprime with said V3 sequence in a gp140 or gp160 boost.
 38. The methodaccording to claim 33 wherein said method comprises administering saidV3 sequence in a DNA prime with said V3 sequence in a gp120, gp140 orgp160 replicating vector boost.
 39. The method according to claim 33wherein said method comprises administering a replicating vectorcomprising said V3 sequence in envelope as prime and boost.